Enzyme-mediated modification of fivrin for tissue engineering

ABSTRACT

The invention provides fibrin-based, biocompatible materials useful in promoting cell growth, wound healing, and tissue regeneration. These materials are provided as part of several cell and tissue scaffolding structures that provide particular application for use in wound-healing and tissue regenerating. Methods for preparing these compositions and using them are also disclosed as part of the invention. A variety of peptides may be used in conjunction with the practice of the invention, in particular, the peptide IKVAV, and variants thereof. Generally, the compositions may be described as comprising a protein network (e.g., fibrin) and a peptide having an amino acid sequence that comprises a transglutaminase substrate domain (e.g., a factor XIIIa substrate domain) and a bioactive factor (e.g., a peptide or protein, such as a polypeptide growth factor), the peptide being covalently bound to the protein network. Other applications of the technology include their use on implantable devices (e.g., vascular graphs), tissue and cell scaffolding. Other applications include use in surgical adhesive or sealant, as well as in peripheral nerve regeneration and angiogenesis.

This application is a divisional of copending application Ser. No.09/057,052, filed Apr. 8, 1998, entitled “ENZYME-MEDIATED MODIFICATIONOF FIBRIN FOR TISSUE ENGINEERING,” by Jeffrey A. Hubbell and JasonSchense now U.S. Pat. No. 6,331,422, which is a continuation to PCTApplication No. PCT/US98/06617, filed Apr. 2, 1998, which claimspriority to U.S. Ser. No. 60/042,143, filed Apr. 3, 1997.

The United States Government has certain rights in this inventionpursuant to Grant No. USPHS HD 31462-01A1, awarded by the NationalInstitute of Health.

BACKGROUND OF THE RELATED ART

Fibrin is a natural gel with several biomedical applications. Fibrin gelhas been used as a sealant because of its ability to bind to manytissues and its natural role in wound healing. Some specificapplications include use as a sealant for vascular graft attachment,heart valve attachment, bone positioning in fractures and tendon repair(Sierra, D. H., Journal of Biomaterials Applications, 7:309-352, 1993).Additionally, these gels have been used as drug delivery devices, andfor neuronal regeneration (Williams, et al., Journal of ComparativeNeurobiology, 264:284-290, 1987).

The process by which fibrinogen is polymerized into fibrin has also beencharacterized. Initially, a protease cleaves the dimeric fibrinogenmolecule at the two symmetric sites. There are several possibleproteases that can cleave fibrinogen, including thrombin, reptilase, andprotease III, and each one severs the protein at a different site(Francis, et al., Blood Cells, 19:291-307, 1993). Each of these cleavagesites have been located (FIG. 1). Once the fibrinogen is cleaved, aself-polymerization step occurs in which the fibrinogen monomers cometogether and form a non-covalently crosslinked polymer gel (Sierra,1993). A schematic representation of the fibrin polymer is shown in FIG.2. This self-assembly happens because binding sites become exposed afterprotease cleavage occurs. Once they are exposed, these binding sites inthe center of the molecule can bind to other sites on the fibrinogenchains, these sites being present at the ends of the peptide chains(Stryer, L. In Biochemistry, W. H. Freeman & Company, N.Y., 1975). Inthis manner, a polymer network is formed. Factor XIIIa, atransglutaminase activated from factor XIII by thrombin proteolysis, maythen covalently cross-link the polymer network. Other transglutaminasesexist and may also be involved in covalent crosslinking and grafting tothe fibrin network.

Once a crosslinked fibrin gel is formed, the subsequent degradation istightly controlled. One of the key molecules in controlling thedegradation of fibrin is α2-plasmin inhibitor (Aoki, N., Progress inCardiovascular Disease, 21:267-286, 1979). This molecule acts bycrosslinking to the α chain of fibrin through the action of factor XIIIa(Sakata, et al., Journal of Clinical Investigation, 65:290-297, 1980).By attaching itself to the gel, a high concentration of inhibitor can belocalized to the gel. The inhibitor then acts by preventing the bindingof plasminogen to fibrin (Aoki, et al., Thrombosis and Haemostasis,39:22-31, 1978) and inactivating plasmin (Aoki, 1979). The α-2 plasmininhibitor contains a glutamine substrate. The exact sequence has beenidentified as NQEQVSPL (SEQ ID NO: 15), with the first glutamine beingthe active amino acid for crosslinking.

The components required for making fibrin gels can be obtained in twoways. One method is to cryoprecipitate the fibrinogen from plasma. Inthis process, factor XIII precipitates with the fibrinogen, so it isalready present. The proteases are purified from plasma using similarmethods. Another technique is to make recombinant forms of theseproteins either in culture or with transgene animals. The advantage ofthis is that the purity is much higher, and the concentrations of eachof these components can be controlled.

Cells interact with their environment through protein-protein,protein-oligosaccharide and protein-polysaccharide interactions at thecell surface. Extracellular matrix proteins provide a host of bioactivesignals to the cell. This dense network is required to support thecells, and many proteins in the matrix have been shown to control celladhesion, spreading, migration and differentiation (Carey, Annual Reviewof Physiology, 53:161-177, 1991). Some of the specific proteins thathave shown to be particularly active include laminin, vitronectin,fibronectin, fibrin, fibrinogen and collagen (Lander, Journal of Trendsin Neurological Science, 12:189-195, 1989). Many studies of laminin havebeen conducted, and it has been shown that laminin plays a vital role inthe development and regeneration of nerves in vivo and nerve cells invitro (Williams, Neurochemical Research, 12:851-869, 1987; Williams, etal., 1993), as well as in angiogenesis.

Some of the specific sequences that directly interact with cellularreceptors and cause either adhesion, spreading or signal transductionhave been identified. This means that the short active peptide sequencescan be used instead of the entire protein for both in vivo and in vitroexperiments. Laminin, a large multidomain protein (Martin, Annual Reviewof Cellular Biology, 3:57-85, 1987), has been shown to consist of threechains with several receptor-binding domains. These receptor-bindingdomains include the YIGSR (SEQ ID NO: 1) sequence of the laminin B1chain ( Graf, et al., Cell, 48:989-996, 1987; Kleinman, et al., Archivesof Biochemistry and Biophysics, 272:39-45, 1989; and Massia, et al., J.of Biol. Chem., 268:8053-8059, 1993), LRGDN (SEQ ID NO: 2) of thelaminin A chain (Ignatius, et al., J. of Cell Biology, 111:709-720,1990) and PDGSR (SEQ ID NO: 3) of the laminin B1 chain (Kleinman, etal., 1989). Several other recognition sequences for neuronal cells havealso been identified. These include IKVAV (SEQ ID NO: 4) of the lamininA chain (Tashiro, et al., J. of Biol. Chem., 264:16174-16182, 1989) andthe sequence RNIAEIIKDI (SEQ ID NO: 5) of the laminin B2 chain (Liesi,et al., FEBS Letters, 244:141-148, 1989). The receptors that bind tothese specific sequences have also often been identified. A subset ofcellular receptors that has shown to be responsible for much of thebinding is the integrin superfamily (Rouslahti, E., J. of Clin.Investigation, 87:1-5, 1991). Integrins are protein heterodimers thatconsist of α and β subunits. Previous work has shown that the tripeptideRGD binds to several β1 and β3 integrins (Hynes, R. O., Cell, 69:1-25,1992; Yamada, K. M., J. of Biol. Chem., 266:12809-12812, 1991),IKVAV(SEQ ID NO: 4) binds to a 110 kDa receptor (Tashiro, et al., J. ofBiol. Chem., 264:16174-16182, 1989; Luckenbill-Edds, et al., Cell TissueResearch, 279:371-377, 1995), YIGSR (SEQ ID NO: 1) binds to a 67 kDareceptor (Graf, et al., 1987) and DGEA (SEQ ID NO: 6), a collagensequence, binds to the α₂,β₁ integrin (Zutter & Santaro, Amer. J. ofPathology, 137:113-120, 1990). The receptor for the RNIAEIIKDI (SEQ IDNO: 5) sequence has not been reported.

Work has been done in crosslinking bioactive peptides to large carriermolecules and incorporating them within fibrin gels. By attaching thepeptides to the large carrier polymers, the rate of diffusion out of thefibrin gel will be slowed down. In one series of experiments,polyacrylic acid was used as the carrier polymer and various sequencesfrom laminin were covalently bound to them to confer neuroactivity(Herbert, C. in Chemical Engineering 146) to the gel. The stability ofsuch a system was poor due to a lack of covalent or high affinitybinding between the fibrin and the bioactive molecule.

Very little work has been done in incorporating peptide sequences andother bioactive factors into fibrin gels and even less has been done incovalently binding peptides directly to fibrin. However, a significantamount of energy has been spent on determining which proteins bind tofibrin via enzymatic activity and often determining the exact sequencewhich binds as well. The sequence for fibrin γ-echain crosslinking hasbeen determined and the exact site has been located as well (Doolittle,et al., Biochem. & Biophys. Res. Comm., 44:94-100, 1971). Factor XIIIahas also been shown to crosslink fibronectin to fibronectin (Barry &Mosher, J. of Biol. Chem., 264:4179-4185, 1989), as well as fibronectinto fibrin itself (Okada, et al., J. of Biol. Chem., 260:1811-1820,1985). This enzyme also crosslinks von Willebrand factor (Hada, et al.,Blood, 68:95-101, 1986), as well as α-2 plasmin inhibitor (Tamaki &Aoki, J. of Biol. Chem., 257:14767-14772, 1982), to fibrin. The specificsequence that binds from α-2 plasmin inhibitor has been isolated(Ichinose, et al., FEBS Letters, 153:369-371, 1983) in addition to thenumber of possible binding sites on the fibrinogen molecule (Sobel &Gawinowicz, J. of Biol. Chem., 271:19288-19297, 1996) for α-2 plasmininhibitor. Thus, many substrates for factor XIIIa exist, and a number ofthese have been identified in detail.

SUMMARY OF THE INVENTION

The present invention in a general and overall sense, provides uniquefusion proteins and other factors, either synthetically orrecombinantly, that contain both a transglutaminase domain such as aFactor XIIIa substrate domain and a bioactive factor, these peptidesbeing covalently attached to a fibrin substrate having athree-dimensional structure capable of supporting cell growth.

In some embodiments of the present invention, bioactive properties foundin extracellular matrix proteins and surface proteins are confined to astructurally favorable matrix that can readily be remodeled bycell-associated proteolytic activity. In some embodiments, the fibrin isgel matrix. A bioactive means is also included to facilitate theincorporation of an exogenous signal into the substrate. In addition toretaining the bioactivity of the exogenous signal molecule, the overallstructural characteristics of the fibrin gel is maintained.

The invention in another aspect provides for a fibrin matrix comprisingshort peptides covalently crosslinked thereto, as well as bioactivefactors. The fibrin matrix may be further defined as a fibrin gel. Thematrix chosen is fibrin, since it provides a suitable three dimensionalstructure for tissue growth and is the native matrix for tissue healing.It is anticipated that other, fibrin-like matrices may also be similarlyprepared. The crosslinking was accomplished enzymatically by using thenative Factor XIIIa to attach the exogenous factors to the gels. Inorder to do this, a sequence that mimics a crosslinking site wasincorporated into the peptide so that the enzyme recognized andcrosslinked it into the matrix. Novel activity will be conferred tothese fibrin gels by adding a peptide sequence, or other bioactivefactor, which is attached to the crosslinking sequence. These materialsmay be useful in the promotion of healing and tissue regeneration, inthe creation of neurovascular beds for cell transplantation and innumerous other aspects of tissue engineering. Hence, the invention inyet other aspects provides compositions created and adapted for thesespecific uses.

The following sequences are referenced throughout the Specification:

SEQ ID NO: DESCRIPTION SEQ ID NO: 1 YIGSR - Peptide that binds to a 67kDa receptor SEQ ID NO: 2 LRGDN - Peptide of the laminin A chain SEQ IDNO: 3 PDGSR - Peptide of the laminin B1 chain SEQ ID NO: 4 IKVAV -Peptide that binds to a 110 kDa receptor SEQ ID NO: 5 RNIAEIIKDI -Peptide of the laminin B2 chain SEQ ID NO: 6 DGEA - A collagen peptidethat binds to the α₂, β₁-integrin SEQ ID NO: 7 PRRARV - A sequence fromfibronectin is also a heparin sulfate binding sequence SEQ ID NO: 8YRGDTIGEGQQHHLGG - A peptide with glutamine at the transglutaminasecoupling site, an active RGD sequence and a dansylated amino acid,mimics the crosslinking site in the γ chain of fibrinogen SEQ ID NO: 9LRGDGAKDV - A peptide that mimics the lysine coupling site in the δchain of fibrinogen with an active RGD sequence and a dansylated leucineadded SEQ ID NO: 10 LRGKKKKG - A peptide with a polylysine at a randomcoupling site attached to an active RGD and a dansylated leucine SEQ IDNO: 11 LNQEQVSPLRGD - A peptide that mimics the crosslinking site in α2-plasmin inhibitor with an active RGD added to the carboxy terminus and adansylated leucine to the amino terminus SEQ ID NO: 12YRGDTIGEGQQHHLGG - A peptide with glutamine at the transglutaminasecoupling site in the chain of fibrinogen SEQ ID NO: 13 GAKDV - A peptidethat mimics the lysine coupling site in the chain of fibrinogen SEQ IDNO: 14 KKKK - A peptide with a polylysine at a random coupling site SEQID NO: 15 NQEQVSPL - A peptide that mimics the crosslinking site in2-plasmin inhibitor SEQ ID NO: 16 LNQEQVSPLGYIGSR - A peptide thatmimics the crosslinking site in α2- plasmin inhibitor with an activeYIGSR added to the carboxy terminus and a dansylated leucine to theamino terminus SEQ ID NO: 17 LNQEQVSPLDDGEAG - A peptide that mimics thecrosslinking site in α2- plasmin inhibitor with an active DGEA SEQ IDNO: 18 LNQEQVSPLRAHAVSE - A peptide that mimics the crosslinking site inα2- plasmin inhibitor with an active HAV added to the carboxy terminusand a dansylated leucine to the amino terminus SEQ ID NO: 19LNQEQVSPRDIKVAVDG - A peptide that mimics the crosslinking site in α2-plasmin inhibitor with an active IKVAV added to the carboxy terminus anda dansylated leucine to the amino terminus SEQ ID NO: 20LNQEQVSPRNIAEIIKDIR - A peptide that mimics the crosslinking site in α2-plasmin inhibitor with an active RNIAEIIKDI added to the carboxyterminus and a daysylated leucine to the amino terminus

In one aspect, the invention provides a composition that comprises aprotein network and a peptide having an amino acid sequence thatcomprises a transglutaminase substrate domain and a bioactive factor(e.g., peptide, protein, or fragment thereof) is provided. The peptideis covalently or at least substantially covalently bound to the proteinnetwork. In particular embodiments, the protein network is fibrin or afibrin-like molecule. In other particular embodiments, thetransglutaminase substrate domain is a factor XIIIa substrate domain.This factor XIII, a substrate domain may be further defined ascomprising an amino acid sequence SEQ ID NO: 12, SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, a fragment thereof, a combination thereof, or abioactive fragment of said combination. Some embodiments may be definedas comprising a bioactive factor that comprises an amino acid sequenceof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, a fragment thereof, a combination thereof, or a bioactivefragment of said combination.

In another aspect, the invention provides an implantable device havingat least one surface or portion of at least one surface that comprisesthe composition of any one of the above compositions described herein.By way of example, the implantable device may be fashioned as anartificial joint device, such as a knee replacement. The invention mayalso take the form of a porous vascular graft, wherein at least oneregion or a portion of at least one region of the porous vascular graftcomprises a porous wall that includes the composition of the proteinnetwork and covalently attached peptide/protein described herein. Theinvention as a device may be further defined in other embodiments as ascaffold for skin, bone, nerve or other cell growth, comprising asurface that includes at least one region or area that comprises thecomposition of the protein matrix and covalently attached peptidedescribed herein.

In yet another aspect, the invention provides for a surgical sealant oradhesive comprising a surface that includes the composition of thepeptide matrix and covalently attached peptide on at least one region ofthe surface.

The invention further provides methods for promoting cell growth ortissue regeneration. This method comprises in some embodiments,covalently attaching or producing a covalently attached bioactivecomplex molecule comprising a bioactive factor and a transglutaminasesubstrate, covalently coupling the bioactive complex molecule to apeptide network capable of having covalently attached thereto thebioactive factor or a fragment thereof, to provide a treated peptidesubstrate; and exposing said treated peptide substrate to a compositioncomprising cells or tissue to promote cell growth or tissueregeneration. This method may be used in conjunction with a variety ofdifferent cell types and tissue types. By way of example, such celltypes include nerve cells, skin cells, and bone cells. The peptidenetwork may be further defined as a protein network, such as a fibrinnetwork. The transglutaminase substrate may be further defined as afactor XIIIa substrate, while the transglutaminase may be furtherdefined as factor XIIIa. The factor XIIIa substrate may be furtherdefined as having an amino acid sequence of SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, a fragment thereof, a combinationthereof, or a bioactive peptide fragment of said composition. Thepeptide may, in some embodiments, be further defined as comprising anamino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ IDNO. 4, SEQ ID NO. 5, SEQ ID NO. 6, a fragment thereof, a combinationthereof or a bioactive peptide fragment thereof.

The invention in yet another aspect may be defined as a biosupportivematrix material. This material in some aspects, comprises a peptidenetwork and a bioactive factor, wherein said bioactive factor iscovalently attached to the peptide substrate. This peptide substrate maybe further defined as a protein network. The bioactive factor iscovalently attached to the substrate through a transglutaminase or asimilar enzyme. The peptide that may be used in conjunction with theinvention may comprise any variety of peptides capable of beingcovalently attached to the fibrin substrate or biosupportive matrix asdescribed herein. In some embodiments, the peptide may be furtherdefined as comprising an amino acid sequence of SEQ ID NO. 1, SEQ ID NO.4, SEQ ID NO. 5, SEQ ID NO. 10, a fragment thereof, a combinationthereof, or a bioactive fragment thereof.

In particular embodiments of the matrix compositions, the calculatedmoles of peptide that is to be included may be defined or described forthose devices/surfaces that include them, as virtually any amount ofpeptide that falls within a physiologically relevant concentration ofthe particular peptide/protein selected. For a standard gel, 1 mg offibrinogen would typically be included. Hence the concentration offibrinogen in this standard gel may be described as about 3×10⁻⁶ mM.Using this figure as a benchmark in one example, the ratio of the amountof peptide to fibrinogen could be expressed as about 3×10⁻⁶ mM to about24×10⁻⁶ mM.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. The homodimeric structure of fibrinogen has been elucidated.Each symmetric half of fibrinogen is itself a heterotrimer of the threechains Am, BE and y. Here, the cleavage sites of the major proteaseshave been marked (R is for reptilase, T is for thrombin and P III is forprotease III). Additionally, some of the sites where cross linking canoccur have been marked as x1.

FIG. 2. A schematic representation of fibrinogen is given. The polymeris held together by the binding of sites B to B′ and A to A′. A′ and Bonly become available for binding after cleavage by a protease. Thepolymerization reaction is self-activated. A single monomer unit isboxed in the center.

FIG. 3. Each curve represents the different crosslinking abilities ofthe four peptides. The molar excess of peptide used is plotted againstthe ratio of peptide molecules to fibrinogen molecules for a series ofpeptide concentrations. Gln and Lys represent the two peptides thatmimic the g-chain of fibrinogen. Polylys is the multiple lysine peptideand pi-1 is the sequence from α2-plasmin inhibitor. In FIG. 3, polylys(SEQ ID NO: 10) is indicated by □, gln (SEQ ID NO: 8) is indicated by ⋄;lys(SEQ ID NO: 9) is indicated by O, and pi-1 (SEQ ID NO: 11) isindicated by Δ.

FIG. 4. Each curve represents the different crosslinking abilities ofthe four peptides. The molar ratio of peptide to fibrinogen in theinitial reaction mixture is varied and plotted on the x axis. The ratioof crosslinked peptide to fibrinogen is then measured and plotted onthey axis. In FIG. 4, dYRGDTIGEGQQHHLGG (SEQ ID NO: 8) is indicated by▪, dLRGDGAKDV (SEQ ID NO: 9) is indicated by ♦, dLNQEQVSPLRGD (SEQ IDNO: 11) is indicated by , and dLRGDKKKKG (SEQ ID NO: 10) is indicatedby ▾.

FIG. 5. Growth normalized against unmodified fibrin RGD; RDG; DGEA;IKVAV (SEQ ID NO: 4); YIGSR (SEQ ID NO: 1); RNIAEIIKDI (SEQ ID NO: 5);and HAV.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

Using standard solid phase peptide synthesis, peptides with sequencesthat combine crosslinking sites from fibrinogen or another protein thatcrosslinks to fibrin gels, and active sequences, such as RGD or IKVAV(SEQ ID NO: 4) were created. A dansyl group was added to the primaryamine of the peptide so that the molecule could be detected when in thepresence of other proteins. The peptides were syringe filtered andfreeze dried to purify.

Fibrin gels were created using thrombin as the enzyme. Thrombin,calcium, dansylated peptide and Tris Buffered Saline (pH 7) were mixedto achieve the proper concentration of all components. Dialyzedfibrinogen that contains residual factor XIII was added and the gelswere polymerized in an incubator. The final gel concentrations for eachcomponent were 4 mg/ml of fibrinogen, 2.5 mM CA⁺⁺, 2 NIH units/mil ofthrombin and various amounts of peptide. The gels were then covered withPhosphate Buffered Saline, and the buffer was changed until all the freepeptide had diffused from the gel. The gels were then degraded with theminimal amount of plasmin necessary to achieve complete degradation.

One method used to analyze the results is as follows. The resultingproducts were run out on a gel permeation chromatography column andanalyzed using a photodiode array detector. With this detector, we cancollect and analyze data at many wavelengths at the same time.Chromatograms of each run were made at 280 nm (this signal isproportional to the total protein present. 205 rim can be used as well).The results were compared to a standard curve created from degradedfibrinogen and the total fibrin concentration was calculated. Afluorescence detector was used to measure the presence of peptide. Thesample was excited at a wavelength of 330 nm and the emitted energy at530 rim was measured (this is proportional to the total amount of dansylgroups present). These results were compared to standard curves createdfor each peptide and the ratio of peptide molecules to fibrin moleculesin the gel was determined for a series of peptide concentrations.Furthermore, since a size exclusion column was used, it could bedetermined if the sizes of the peptide fragments in the gel were larger,smaller or the same as that of free peptide. If they are larger, thenthis is evidence that the peptide is directly bound to some fragment ofgel and a covalent bond has actually been formed.

A second method used to analyze the substrates of the present inventionfor amount of peptide was as follows. Each gel was washed several times,and the amount of peptide present in each wash was measured on aspectofluorimeter. The gels were then degraded with plasmin and then theamount of fluor present was measured. The percent of fluor in the gelcompared to the washes was calculated and since the initial peptide massis known, the mass of peptide in the gels was calculated from this. Whenthe fibrinogen was dissolved, the total mass dissolved was known andthis was used to determine the mass of fibrinogen present in the gel. Adifferent concentration of peptide was used in each series of studiesand curves relating the total peptide incorporated with the initialpeptide used were made.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 PEPTIDE BOUND PER MOLECULE OF FIBRINOGEN TO FIBRIN GELS

By washing peptide decorated gels, degrading them with plasmin andperforming size exclusion chromatography, the amount of peptide boundper molecule of fibrinogen was calculated for a series of peptideconcentrations and for four separate peptide sequences. All thesubstrate sequences tested included RGD as an exemplary bioactivesequence. The sequences tested include two that mimic the crosslinkingsite in the δ chain of fibrinogen, *YRGDTIGEGQQHHLGG (SEQ ID NO: 8) (*indicates the dansyl group and the section in italics is the nativesequence of the crosslinking region of fibrinogen), a peptide withglutamine at the transglutaminase coupling site, and *LRGDGAKDV(SEQ IDNO: 9), a mimic of the lysine coupling site. Additionally a peptide witha polylysine at a random coupling site, *LRGDKKKKG (SEQ ID NO: 10), anda sequence that mimics the crosslinking site in α 2-plasmin inhibitor,*LNQEQVSPLRGD (SEQ ID NO: 11) were also used. The amount of peptidecovalently bound to the fibrin gels was measured while varying theinitial excess of peptide for each of the four sequences. Aconcentration dependent curve was created (FIG. 3) and the maximumcrosslinking ratio and the molar excess needed to achieve a 1:1 ratioare shown below in Table 1. Since a particular active sequence isusually present once in each protein, the excess of peptide required toachieve this concentration provides an interesting benchmark. Thepeptide that provides the greatest possible crosslinking concentrationwill provide the most flexibility. From the results seen in FIG. 4, theplasmin inhibitor peptide is the best, since it provides the highestcrosslinking concentration and the greatest crosslinking efficiency.

TABLE 1 Maximum Molar excess Crosslinking Ratio needed to achievePeptide Sequence Pep/Fibrinogen 1:1 ratio *YRGDTIGEGQQHHLGG 1.53 12 SEQID NO: 8 *LRGDGAKDV 0.44 >330 SEQ ID NO: 9 *LRGDKKKKG 1.2 11 SEQ ID NO:10 *LNQEQVSPLRGD 8.2 6 SEQ ID NO: 11

This table shows the amount of peptide needed to covalently bind onepeptide molecule per fibrinogen molecule in a fibrin gel.

A collection of peptides utilizing the crosslinking sequence fromα2-plasmin inhibitor have been made using active peptide sequences fromthe basement membrane molecules laminin and collagen SEQ ID NO: 11, and16-20). Eight day chicken dorsal root ganglia were polymerized insidegels that had enough peptide to achieve the highest crosslinkedconcentration possible (8 moles peptide/mole fibrinogen). The extensionof neurites from the ganglia was measured at 24 and 48 hours. The 48hour data is shown in FIG. 5. The average neurite length for eachexperimental condition was normalized against growth in unmodifiedfibrin. Four of the active peptides used, IKVAV (SEQ ID NO: 4),RNIAEIIKDI (SEQ ID NO: 5), YIGSR (SEQ ID NO: 1) and RGD demonstratedstatistically different neurine growth, proving that not only candifferent factors be attached to the fibrin gels, but they retainbiologically significant activity. Soluble inhibitor experiments werecompleted as well, and in each trial, the neurite growth wasstatistically the same as unmodified fibrin. This result demonstratesthat the activity is interrupted, then the presence of crosslinkedpeptide does not inhibit neural extension. the growth in RDG crosslinkedfibrin also supports this conclusion, as the neurites are able to attainsimilar growth with this nonactive peptide presence as is achieved inunmodified fibrin.

EXAMPLE 2

The present example is provided to demonsrate the utility of the presentinvention for providing the covalent attachment of a bioactive factor toa peptide matrix, the amount of the bioactive factor, such as a peptide,being quantitatively determinable.

Using the spectrofluorimetry method (second method) described above, theamount of peptide bound per molecule of fibrinogen was calculated for aseries of peptide concentrations and for four separate peptidesequences. The sequences tested include two that mimic the crosslinkingsite in the γ chain of fibrinogen, *YRGDTIGEGQQHHLGG (SEQ ID NO: 8) (*indicates the dansyl group and the section in italics is the nativesequence of the crosslinking region of fibrinogen), a peptide withglutamine at the transglutaminase coupling site, and *LRGDGAKDV(SEQ IDNO: 9), a mimic of the lysine coupling site. Additionally a peptide witha polylysine at a random coupling site, *LRGDKKKKG (SEQ ID NO: 10), anda sequence that mimics the crosslinking site in α2-plasmin inhibitor,*LNQEQVSPLRGD (SEQ ID NO: 11) were also tested. The coupling of eachpeptide used was measured by determining the excess moles of peptideneeded to get one peptide covalently bound to each fibrinogen moleculepresent. Since a particular active sequence is usually present once ineach protein, this is a suitable benchmark. From the results seen inFIG. 3, it is clear that the plasmin inhibitor peptide (pi-1) is thebest, the peptide with the sequence of multiple lysines (polylys) hasthe second highest coupling rate, while the two γ chain peptides (glnand lys) follow. The actual amount of peptide needed to achieve a 1:1ratio of peptide to fibrinogen is shown in Table 2.

TABLE 2 Peptide sequence Molar excess needed to achieve 1:1 ratio*YRGDTIGEGQQHHLGG 110 SEQ ID NO: 8 *LRGDGAKDV 220 SEQ ID NO: 9 LRGDKKKKG39 SEQ ID NO: 10 *LNQEQVSPLRGD ˜10 SEQ ID NO: 11

Table 2 shows the amount of peptide needed to covalently bind onepeptide molecule per fibrinogen molecule in a fibrin gel.

A Factor XIIIa substrate has been synthetically coupled to a bioactivepeptide sought for incorporation into the fibrin matrix, and it is clearthat this bioactive factor need not have been a peptide. While notintending to be limited to any particular mechanism of action or theoryof operation, any bioactive or biologically or medically useful moleculeor macromolecule could be the bioactive factor. Likewise, the couplingbetween the bioactive factor and the transglutaminase substrate domaincould have been performed by recombinant DNA methodology or any othermeans. For example, a protein growth factor could be incorporated byrecombinantly expressing a fusion protein comprising both atransglutaminase substrate domain and the growth factor domain.Furthermore, the transglutaminase substrate domain could be targeted fora translutaminase other than facor XIIIa. Furthermore, a recombinantform of fibrinogen could be used to form the fibrin network.Furthermore, other proteins that transglutaminase recognizes, such asfibronectin for example, could be coupled to the transglutaninasesubstrate peptide.

There are numerous applications for these fibrin gels that arederivitized with a bioactive factor. Fibrin is a natural matrix found inthe body and is utilized in many ways. Although fibrin does provide asolid support for tissue regeneration and cell ingrowth, there are fewactive sequences in the monomer that directly enhance these processes.However, other studies have shown that many proteins, including basementmembrane proteins such as laminin and growth factors such as basicfibroblast growth factor, have sequences which directly enhanceregeneration or migration. Our method allows us to incorporate an activesequence or entire factor into the gels and create gels which possessspecific bioactive properties.

The present invention provides the first description of a means by whichto effectively incorporate bioactive factors into fibrin, atherapeutically important material in wound healing and tissueengineering have been provided. Hence a previously unaccomplished goalis presented that provides an important therapeutic material.

EXAMPLE 3 BIOACTIVITY IN SITU GANGLIA MODEL

Bioactivity can be quantified using cell studies based on the 8-daychicken dorsal root ganglia model. With this model, addition ofneuronally active sequences to the peptide can be tested for theirability in vitro to enhance neurite extension. Ganglia were dissectedfrom eight day old chicken embryos and fibrin gels were polymerizedaround them. Peptide with different active sequences was crosslinkedinto these gels and the unbound peptide was washed out by periodicallychanging the neuronal media on top of the gels. These ganglia thenextend neurites in three dimensions and the projection of these neuritescan be captured using imaging software. This image can then be used tocalculate the average neurite length. Three control experiments weredone. Neurites were grown in fibrin gels without any peptidecrosslinked, in fibrin gels with a nonactive peptide crosslinked in andin gels with active peptide crosslinked and soluble peptide present inthe media as an inhibitor.

EXAMPLE 4 NERVE REGENERATION AND SCAFFOLD

The present example demonstrates the utility of the present invention asa tissue regenerational supportive material. In addition, the data heredemonstrates the utility of the invention for supporting the effectiveregeneration of nerve tissue.

A collection of peptides utilizing the crosslinking sequence fromα2-plasmin inhibitor have been made using active peptide sequences fromthe basement membrane molecules laminin and collagen. Eight day chickendorsal root ganglia were polymerized inside gels that had enough peptideto achieve the highest crosslinked concentration possible (8 molespeptide/mole fibrinogen). The extension of neurites from the ganglia wasmeasured at 24 and 48 hours. The 48 hour data is shown in FIG. 5. Theaverage neurite length for each experimental condition was normalizedagainst growth in unmodified fibrin. Four of the active peptides used,IKVAV (SEQ ID NO: 4), RNIAEIIKDI (SEQ ID NO: 5), YIGSR (SEQ ID NO: 1)and RGD, demonstrated statistically different neurite growth, provingthat not only can different factors be attached to the fibrin gels, butthey retain biologically significant activity. Soluble inhibitorexperiments were completed as well, and in each trial, the neuritegrowth was statistically the same as unmodified fibrin. This resultdemonstrates that the activity of each sequence added is dependant onthe physical crosslinking. Furthermore, this shows that if the neuronalactivity of the attached factor is interrupted, then the presence ofcrosslinked peptide does not inhibit neural extension. The growth in RDGcrosslinked fibrin also supports this conclusion, as the neurites areable to attain similar growth with this nonactive peptide present as isachieved in unmodified fibrin.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references are specifically incorporated herein byreference for the various purposes described herein.

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3. Francis, et al., “Endothelial Cell Responses to Fibrin Mediated byFPB Cleavage and the Amino Terminus of the B Chain”; Blood Cells,19:291-307, 1993.

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7. Aoki, et al., “Effects of α 2-plasmin inhibitor on fibrin clot lysis.Its comparison with α 2-macroglobulin,.” Thrombosis and Haemostasis,39:22-31, 1978.

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9. Lander, A., “Understanding the molecules of cell contacts,” Journalof Trends in Neurological Science, 12:189-195, 1989.

10. Williams, L. R., “Exogenous fibrin matrix precursors stimulate thetemporal progress of nerve regeneration within a silicone chamber,”Neurochemical Research, 12:851-860, 1987.

11. Martin, G. R., “Laminin and other basement membrane proteins,”Annual Review of Cellular Biology, 3:57-85, 1987.

12. Graf, et al., “Identification of an Amino Acid Sequence in LamininMediating Cell Attachment, Chemotaxis, and Receptor Binding,” Cell,48:989-996, 1987.

13. Kleinman, et al., “Identification of a second site in laminin forpromotion of cell adhesion and migration and inhibition of in vivomelanoma lung colonization,” Archives of Biochemistry and Biophysics,272:39-45, 1989.

14. Massia, et al., “Covalently immobilized laminin peptidetyr-ile-gly-ser-arg (YIGSR) supports cell spreading and colocalizationof the 67 kilodalton receptor with α-actinin and viniculin,” Journal ofBiological Chemistry, 268:8053-8059, 1993.

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21. Tashiro, et al., “A synthetic peptide containing the IKVAV sequencefrom a chain of laminin mediates cell attachment, migration and neuriteoutgrowth,” Journal of Biological Chemistry, 264:16174-16182, 1989.

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20 5 amino acids amino acid <Unknown> linear 1 Tyr Ile Gly Ser Arg 1 5 5amino acids amino acid <Unknown> linear 2 Leu Arg Gly Asp Asn 1 5 5amino acids amino acid <Unknown> linear 3 Pro Asp Gly Ser Arg 1 5 5amino acids amino acid <Unknown> linear 4 Ile Lys Val Ala Val 1 5 10amino acids amino acid <Unknown> linear 5 Arg Asn Ile Ala Glu Ile IleLys Asp Ile 1 5 10 4 amino acids amino acid <Unknown> linear 6 Asp GlyGlu Ala 1 6 amino acids amino acid <Unknown> linear 7 Pro Arg Arg AlaArg Val 1 5 16 amino acids amino acid <Unknown> linear 8 Tyr Arg Gly AspThr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly 1 5 10 15 9 amino acidsamino acid <Unknown> linear 9 Leu Arg Gly Asp Gly Ala Lys Asp Val 1 5 9amino acids amino acid <Unknown> linear 10 Leu Arg Gly Asp Lys Lys LysLys Gly 1 5 12 amino acids amino acid <Unknown> linear 11 Leu Asn GlnGlu Gln Val Ser Pro Leu Arg Gly Asp 1 5 10 16 amino acids amino acid<Unknown> linear 12 Tyr Arg Gly Asp Thr Ile Gly Glu Gly Gln Gln His HisLeu Gly Gly 1 5 10 15 5 amino acids amino acid <Unknown> linear 13 GlyAla Lys Asp Val 1 5 4 amino acids amino acid <Unknown> linear 14 Lys LysLys Lys 1 8 amino acids amino acid <Unknown> linear 15 Asn Gln Glu GlnVal Ser Pro Leu 1 5 15 amino acids amino acid <Unknown> linear 16 LeuAsn Gln Glu Gln Val Ser Pro Leu Gly Tyr Ile Gly Ser Arg 1 5 10 15 15amino acids amino acid <Unknown> linear 17 Leu Asn Gln Glu Gln Val SerPro Leu Asp Asp Gly Glu Ala Gly 1 5 10 15 16 amino acids amino acid<Unknown> linear 18 Leu Asn Gln Glu Gln Val Ser Pro Leu Arg Ala His AlaVal Ser Glu 1 5 10 15 17 amino acids amino acid <Unknown> linear 19 LeuAsn Gln Glu Gln Val Ser Pro Arg Asp Ile Lys Val Ala Val Asp 1 5 10 15Gly 19 amino acids amino acid <Unknown> linear 20 Leu Asn Gln Glu GlnVal Ser Pro Arg Asn Ile Ala Glu Ile Ile Lys 1 5 10 15 Asp Ile Arg

What is claimed is:
 1. A composition comprising a fibrin network and apeptide having an amino acid sequence that comprises onetransglutaminase substrate domain and a bioactive factor, wherein thepeptide is covalently bound to the fibrin network by thetransglutaminase substrate domain.
 2. The composition of claim 1 whereinthe transglutaminase substrate domain is a factor XIIIa substratedomain.
 3. The composition of claim 2 wherein the factor XIIIa substratedomain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, and combinations and bioactive fragments thereof.
 4. The compositionof claim 2 wherein the factor XIIIa substrate domain comprises an aminoacid sequence of SEQ ID NO:
 15. 5. The composition of claim 1 whereinthe bioactive factor is a peptide.
 6. The composition of claim 1 whereinthe wherein the bioactive factor comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and combinations andbioactive fragments thereof.
 7. The composition of claim 1 wherein thebioactive factor is a protein.
 8. The composition of claim 1 wherein thebioactive factor is a polypeptide growth factor.
 9. A method forpromoting cell growth or tissue regeneration comprising: covalentlyattaching a bioactive factor to a transglutaminase substrate to form abioactive complex molecule; covalently coupling the bioactive complexmolecule to a fibrin network to form a treated fibrin substrate; andexposing the treated fibrin substrate to a composition comprising cellsor tissue to promote cell growth or tissue regeneration.
 10. The methodof claim 9 wherein the composition comprising cells comprises nervecells.
 11. The method of claim 9 wherein the composition comprisingcells comprises skin cells.
 12. The method of claim 9 wherein thetransglutaminase substrate is a factor XIIIa substrate.
 13. The methodof claim 12 wherein the factor XIIIa substrate comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, and combinations and bioactivefragments thereof.
 14. The method of claim 13 wherein the factor XIIIasubstrate comprises an amino acid sequence of SEQ ID NO:
 15. 15. Themethod of claim 9 wherein the bioactive factor is a peptide.
 16. Themethod of claim 9 wherein the bioactive factor is a protein.
 17. Themethod of claim 9 wherein the bioactive factor is a polypeptide growthfactor.
 18. The method of claim 9 wherein the bioactive factor comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,and combinations and bioactive fragments thereof.